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• W2034856955 abstract "The human type II keratin 6 (K6; 56 kDa) is expressed in a heterogeneous array of epithelial tissues under normal conditions, but is better known for its strong induction in stratified epithelia that feature an enhanced cell proliferation rate or abnormal differentiation. Previous work has established the existence of two functional genes encoding K6 protein isoforms in the human genome, although only a partial cDNA clone is available for K6a, the dominant human K6 isoform in skin epithelial tissues (Tyner, A., and Fuchs, E. (1986) J. Cell Biol. 103, 1945-1955). We screened human genomic and skin cDNA libraries with probes derived from the K6b gene, and isolated clones containing the full-length gene and cDNA predicted to encode K6a. A thorough characterization of a large number of genomic (57) as well as cDNA (64) clones further revealed the existence of as many as six different human K6 protein isoforms that are highly related at the gene structure, nucleotide sequence, and predicted amino acid sequence levels. Based on the information accumulated to date we propose an evolutionary model in which the multiplicity of human K6 genes is explained by successive gene duplication events. We further demonstrate that K6a is clearly the dominant K6 isoform in skin tissue samples and cultured epithelial cell lines and that the various isoforms are differentially regulated within and between epithelial tissue types. Our findings have direct implications for an understanding of the regulation and function of K6 during hyperproliferation in stratified epithelia and the search for disease-causing mutations in K6 sequences in the human population. The human type II keratin 6 (K6; 56 kDa) is expressed in a heterogeneous array of epithelial tissues under normal conditions, but is better known for its strong induction in stratified epithelia that feature an enhanced cell proliferation rate or abnormal differentiation. Previous work has established the existence of two functional genes encoding K6 protein isoforms in the human genome, although only a partial cDNA clone is available for K6a, the dominant human K6 isoform in skin epithelial tissues (Tyner, A., and Fuchs, E. (1986) J. Cell Biol. 103, 1945-1955). We screened human genomic and skin cDNA libraries with probes derived from the K6b gene, and isolated clones containing the full-length gene and cDNA predicted to encode K6a. A thorough characterization of a large number of genomic (57) as well as cDNA (64) clones further revealed the existence of as many as six different human K6 protein isoforms that are highly related at the gene structure, nucleotide sequence, and predicted amino acid sequence levels. Based on the information accumulated to date we propose an evolutionary model in which the multiplicity of human K6 genes is explained by successive gene duplication events. We further demonstrate that K6a is clearly the dominant K6 isoform in skin tissue samples and cultured epithelial cell lines and that the various isoforms are differentially regulated within and between epithelial tissue types. Our findings have direct implications for an understanding of the regulation and function of K6 during hyperproliferation in stratified epithelia and the search for disease-causing mutations in K6 sequences in the human population. INTRODUCTIONKeratins are epithelial-specific intermediate filament (IF) 1The abbreviations used are: IFintermediate filamentKkeratinbpbase pairkbpkilobase pairPCRpolymerase chain reaction. proteins encoded by a large multigene family. The ~25 keratins (molecular mass 40-70 kDa) expressed in “soft” epithelial tissues (excluding hair and nail) have been subdivided into type I (K9-K20) and type II (K1-K8) IF sequences(1Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4495) Google Scholar, 2Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1272) Google Scholar). As keratin filament assembly begins with the formation of a type I-type II heterodimer(3Coulombe P.A. Curr. Opin. Cell Biol. 1993; 5: 17-29Crossref PubMed Scopus (95) Google Scholar), epithelial cells express at least one member of each subtype. Pairwise keratin gene expression is regulated in an epithelial tissue-type and differentiation-specific manner, creating patterns that have been well conserved among mammalian species(1Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4495) Google Scholar, 4O'Guin W.M. Schermer A. Lynch M. Sun T-T. Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Publishing Corp., New York1990: 301-334Crossref Google Scholar). In stratified epithelia, the type II K5 and type I K14 genes are transcriptionally active in mitotically active basal cells (5Fuchs E. J. Cell Sci. Suppl. 1993; 17: 197-208Crossref PubMed Google Scholar, 6Schweizer J. Molecular Biology of the Skin: The Keratinocyte. Academic Press, New York1993: 33-78Google Scholar), while other pairs of keratin genes are transcribed in the differentiating cell layers. In epidermis, the main differentiation-specific keratins are K1 and K10, while in esophagus and cornea, they are K4 and K13, and K3 and K12, respectively(4O'Guin W.M. Schermer A. Lynch M. Sun T-T. Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Publishing Corp., New York1990: 301-334Crossref Google Scholar, 5Fuchs E. J. Cell Sci. Suppl. 1993; 17: 197-208Crossref PubMed Google Scholar, 6Schweizer J. Molecular Biology of the Skin: The Keratinocyte. Academic Press, New York1993: 33-78Google Scholar, 7Schermer A. Jester J.V. Hardy C. Milano D. Sun T.T. Differentiation. 1989; 42: 103-110Crossref PubMed Scopus (97) Google Scholar). These keratin pairs appear to be specific for the program of terminal differentiation executed in these tissues(4O'Guin W.M. Schermer A. Lynch M. Sun T-T. Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Publishing Corp., New York1990: 301-334Crossref Google Scholar). In the cytoplasm of epidermal cells, the primary function of keratin filaments is to provide the strength necessary to maintain integrity when skin is subjected to mechanical stress. Alterations in the structure of keratin filaments at any level within the epidermis causes it to rupture within the cell layer(s) affected upon mild mechanical trauma(3Coulombe P.A. Curr. Opin. Cell Biol. 1993; 5: 17-29Crossref PubMed Scopus (95) Google Scholar, 8Fuchs E. Coulombe P.A. Cheng J. Chan T-M. Hutton E. Syder A. Degenstein L. Yu Q-C Letai A. Vassar R. J. Invest. Dermatol. 1994; 103: 25S-30SAbstract Full Text PDF PubMed Scopus (72) Google Scholar). The production of such phenotypes through the directed expression of mutant keratins in the skin of transgenic mice paved the way for the discovery of mutations affecting specific keratins in individuals suffering from a variety of genodermatoses featuring trauma-induced blistering of the epidermis(8Fuchs E. Coulombe P.A. Cheng J. Chan T-M. Hutton E. Syder A. Degenstein L. Yu Q-C Letai A. Vassar R. J. Invest. Dermatol. 1994; 103: 25S-30SAbstract Full Text PDF PubMed Scopus (72) Google Scholar, 9Lane E.B. Curr. Opin. Genet. Dev. 1994; 4: 412-418Crossref PubMed Scopus (42) Google Scholar, 10Steinert P.M. Bale S.J. Trends Genet. 1993; 9: 280-284Abstract Full Text PDF PubMed Scopus (49) Google Scholar).The type II keratin 6 (K6; 56 kDa) is remarkable by several criteria. In contrast to many other keratins, the pairwise expression of K6 and its type I partners K16 and/or K17 is not linked with a well defined program of terminal differentiation(1Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4495) Google Scholar, 4O'Guin W.M. Schermer A. Lynch M. Sun T-T. Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Publishing Corp., New York1990: 301-334Crossref Google Scholar). Thus, K6 is constitutively expressed in distinct types of epithelia, such as filiform papillae of tongue, several “wet” stratified epithelia lining the oral mucosa and esophagus, the outer root sheath of hair follicles, and in glandular epithelia(11Moll R. Moll I. Wiest W. Differentiation. 1982; 23: 170-178Crossref PubMed Scopus (181) Google Scholar, 12Moll R. Krepler R. Franke W.W. Differentiation. 1983; 23: 256-269Crossref PubMed Scopus (307) Google Scholar, 13Ouhayoun J. Gosselin F. Forest N. Winter S. Franke W.W. Differentiation. 1985; 30: 123-129Crossref PubMed Scopus (104) Google Scholar, 14Stark H.J. Breikreutz D. Limat A. Bowden P. Fusenig N.E. Differentiation. 1987; 35: 236-248Crossref PubMed Scopus (115) Google Scholar). With the exception of specific body sites, e.g. palm and sole, K6 is not expressed in epidermis unless it undergoes enhanced proliferation or abnormal differentiation (15Weiss R.A. Eichner R. Sun T.T. J. Cell Biol. 1984; 98: 1397-1406Crossref PubMed Scopus (447) Google Scholar, 16Tyner A.L. Fuchs E. J. Cell Biol. 1986; 103: 1945-1955Crossref PubMed Scopus (123) Google Scholar, 17Stoler A. Kopan R. Duvic M. Fuchs E. J. Cell Biol. 1988; 107: 427-446Crossref PubMed Scopus (292) Google Scholar). K6 and K16 are in fact best known for their induction in epidermis and other stratified epithelia undergoing hyperproliferation, e.g. during wound healing, in several diseases (e.g. psoriasis, actinic keratosis) and in cancer(12Moll R. Krepler R. Franke W.W. Differentiation. 1983; 23: 256-269Crossref PubMed Scopus (307) Google Scholar, 15Weiss R.A. Eichner R. Sun T.T. J. Cell Biol. 1984; 98: 1397-1406Crossref PubMed Scopus (447) Google Scholar, 17Stoler A. Kopan R. Duvic M. Fuchs E. J. Cell Biol. 1988; 107: 427-446Crossref PubMed Scopus (292) Google Scholar). Likewise, K6 induction also occurs when epidermal, corneal, and tracheal cells are seeded in primary culture in vitro(4O'Guin W.M. Schermer A. Lynch M. Sun T-T. Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Publishing Corp., New York1990: 301-334Crossref Google Scholar, 7Schermer A. Jester J.V. Hardy C. Milano D. Sun T.T. Differentiation. 1989; 42: 103-110Crossref PubMed Scopus (97) Google Scholar, 15Weiss R.A. Eichner R. Sun T.T. J. Cell Biol. 1984; 98: 1397-1406Crossref PubMed Scopus (447) Google Scholar, 18Kopan R. Fuchs E. J. Cell Biol. 1989; 109: 295-307Crossref PubMed Scopus (117) Google Scholar). The association between a faster cell turnover rate and K6 expression in stratified epithelia is intriguing, given that expression occurs post-mitotically and can be uncoupled from mitosis in cultured keratinocytes(7Schermer A. Jester J.V. Hardy C. Milano D. Sun T.T. Differentiation. 1989; 42: 103-110Crossref PubMed Scopus (97) Google Scholar, 18Kopan R. Fuchs E. J. Cell Biol. 1989; 109: 295-307Crossref PubMed Scopus (117) Google Scholar). The function(s) that K6 may play in stratified epithelia displaying an enhanced mitotic activity and during wound healing remain to be defined.Another characteristic of K6 is the existence of two functional genes encoding highly related protein isoforms in the human(16Tyner A.L. Fuchs E. J. Cell Biol. 1986; 103: 1945-1955Crossref PubMed Scopus (123) Google Scholar), bovine (19Blessing M. Zentgraf H. Jorcano J.L. EMBO J. 1987; 6: 567-575Crossref PubMed Scopus (72) Google Scholar), and mouse (20Rothnagel J.A. Seki T. Longley M.A. Holder R. Bundman D. Adachi K. Roop D.R. J. Invest. Dermatol. 1993; 101 (Abstr. 235): 563Crossref Scopus (56) Google Scholar) genomes. Of the two known human K6 isoform genes, K6a is more abundantly expressed than K6b at the mRNA level in skin explant cultures(21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar). While the human K6b gene has been characterized (21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar), only a partial cDNA is available for human K6a(22Hanukoglu I. Fuchs E. Cell. 1983; 33: 915-924Abstract Full Text PDF PubMed Scopus (202) Google Scholar). As part of our efforts to understand the role(s) of K6 in hyperproliferative stratified epithelia, we cloned and characterized the human K6a gene and cDNA. While doing so, we discovered the existence of hitherto unknown functional K6 genes in the human genome. The K6 genes and cDNAs we isolated are predicted to encode five or six highly related K6 isoforms, and are differentially regulated in several epithelial cells and tissues examined. We are also proposing a model for the evolution of human K6 genes. While the functional consequences of this remarkable sequence multiplicity have yet to be determined, our findings have direct implications for an understanding of the function of K6 during wound healing as well as the search for point mutations in K6 sequences in the human population.EXPERIMENTAL PROCEDURESMaterialsMaterials were obtained from the following sources: λDashII, λZapII, pBluescript vector, and Gigapack Gold II from Stratagene; restriction endonucleases and DNA modifying enzymes from New England Biolabs; Moloney murine leukemia virus, and Superscript II reverse transcriptases from Life Technologies, Inc.; oligo(dT)-Latex from Roche; Nytran from Schleicher & Schuell; Biodyne nylon membrane from Pall Ultrafine Filtration Corp.; GeneScreen Plus from Dupont. Cell culture medium, fetal bovine serum, glutamine, and antibiotics were purchased from BioWhittaker. All other chemicals were of reagent grade.Human Tissues and Construction of LibrariesDNA was extracted from human placenta and used for the construction of a genomic library. A skin cDNA library was constructed from poly(A) mRNAs extracted from a squamous cell carcinoma of the lower leg (with adjacent normal tissue) obtained from a patient (excision surgery). Other human tissues were obtained as discarded material in the course of surgery or at autopsy.Cell CultureSCC-13, a human skin squamous cell carcinoma line(23Wu Y.J. Parker L.M. Binder N.E. Beckett M.A. Sinard J.H. Griffiths C.T. Rheinwald J.G. Cell. 1982; 31: 693-703Abstract Full Text PDF PubMed Scopus (389) Google Scholar), was grown on a NIH 3T3 fibroblast feeder layer in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum, 0.4 μg/ml hydrocortisone, and 10 ng/ml epidermal growth factor. SCC-4 and SCC-9, two human tongue squamous cell carcinoma lines (23Wu Y.J. Parker L.M. Binder N.E. Beckett M.A. Sinard J.H. Griffiths C.T. Rheinwald J.G. Cell. 1982; 31: 693-703Abstract Full Text PDF PubMed Scopus (389) Google Scholar), were grown in 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's media supplemented with 10% fetal bovine serum and 0.4 μg/ml hydrocortisone. PtK-2, a kangaroo rat kidney cell line(24Franke W.W. Schmid E. Osborn M. Weber K. Cytobiologie. 1978; 17: 391-411Google Scholar), was grown in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, non-essential amino acids, and sodium pyruvate. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.Human Genomic DNA Library ScreeningWe used a human genomic library constructed in the λDashII cloning vector (25Takahashi K. Tanaka A. Hara M. Nakanishi S. Eur. J. Biochem. 1992; 204: 1025-1033Crossref PubMed Scopus (111) Google Scholar) to screen for human K6 genes. Approximately 1.5 × 106 phage clones were screened with DNA probes radiolabeled with [α-32P]dCTP. The initial screening was done using three probes on replicated filters: a 483-bp ApaI fragment derived from exon 1 and a 286-bp RsaI-SacI fragment from the 3′ non-coding region of the cloned human K6b gene(21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar); and a 216-bp AluI-SpeI fragment from the 3′ non-coding region of the cloned human partial K6a cDNA(22Hanukoglu I. Fuchs E. Cell. 1983; 33: 915-924Abstract Full Text PDF PubMed Scopus (202) Google Scholar). Hybridization was carried out under stringent conditions at 65°C in 1 M NaCl, 10% dextran sulfate, and 1% SDS. Filter washing was performed at 65°C in 0.1 × saline sodium citrate (SSC; 150 mM NaCl and 15 mM sodium citrate). Hybridization-positive clones were isolated by repeated plaque purification using standard procedures(26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNAs extracted from the purified phage clones were analyzed by restriction digests and Southern blotting using probes corresponding to one or several exons of the human K6b gene. DNA fragments expected to contain an exon or exons on the basis of blot-hybridization analysis were isolated and subcloned into pBluescript SK(+). DNA sequencing was carried out as described (27Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52361) Google Scholar). Nucleotide and amino acid sequences were compared using the DNASIS-Mac 2.0 software using the simple homology routine (Hitachi Software Engineering Co.).Human Skin cDNA Library ScreeningTo obtain human K6 cDNA clones, we screened a human cDNA library constructed using poly(A) RNA extracted from a leg skin squamous cell carcinoma. After oligo(dT)-driven reverse transcription and second-strand cDNA synthesis, EcoRI adapters were ligated, and fractions containing cDNAs of 2-5 kb were inserted into a λZap II vector. A total of 1.4 × 106 cDNA clones were screened at high stringency (0.1 × SSC, 65°C) with two different probes derived from exon 1 of the human K6a gene isolated in this study (a ~450-bp XhoI-NarI and a ~500-bp ApaI fragment). Positive cDNA clones were further analyzed by oligonucleotide hybridization, PCR, and DNA sequencing. Oligonucleotide hybridization was used to discriminate among K6 isoform cDNAs on the basis of codon 155 sequence (encoding either Ala or Thr, depending on the isoform). The antisense oligonucleotides used were: probe A (Table 1), to detect the K6a isoform (Thr155); and probe B (Table 1), to detect all other K6 isoforms (Ala155; see “Results”). Isolated K6 cDNA clones were blotted onto nylon membrane, and hybridization with probes A or B was carried out at 37°C in 5 × SSC, 30% formamide, and washed in a 0.5 × SSC, 0.5% SDS solution at 60°C. cDNA clones hybridizing positively with probe A or B were purified and rescued into pBluescript SK(-) for analysis.Tabled 1 Open table in a new tab Northern Blot Analysis and Primer-extension AnalysesRNAs were isolated from primary cultures of human skin keratinocytes and several human epithelial carcinoma cell lines by the acid-phenol extraction method(28Bessho Y. Nakanishi S. Nawa H. Brain Res. Mol. Brain Res. 1993; 18: 201-208Crossref PubMed Scopus (56) Google Scholar). These RNA samples were subjected to Northern analysis using genomic DNA fragments from the 3′ non-coding sequence of the K6a and K6b genes as probes. For primer-extension analysis of the K6a mRNA, we used an oligodeoxynucleotide primer specific for the 5′ leader sequence of the K6a mRNA (K6a in Table 1). The primer was labeled with 32P at its 5′ end (2.0-5.0 × 104 cpm), hybridized to total RNA (20 μg) for 4 h at 30°C, and the mixture was subjected to reverse transcription as described previously (25Takahashi K. Tanaka A. Hara M. Nakanishi S. Eur. J. Biochem. 1992; 204: 1025-1033Crossref PubMed Scopus (111) Google Scholar). The primer-extended products were electrophoresed along with the corresponding sequencing reaction on a 7 M urea, 6% polyacrylamide gel.Quantitation of K6 Isoform mRNA Levels by a Colony Hybridization AssaymRNAs were extracted from various human tissues and epithelial cell lines (see above), primed with oligo(dT), and cDNA synthesis was carried out in vitro(26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Small aliquots of cDNA products were used for PCR reactions (94°C for 40 s; 54°C for 40 s; 72°C for 60 s; total of 30 cycles) with a set of universal K6 primers, 5′ K6-primer, and 3′ K6-primer (Table 1). The target sequences of these two primers are perfectly conserved among the K6 isoform genes isolated (K6 a, b, c, d) and cDNAs (K6 a, b, e, f), and amplify a 636-bp long fragment encompassing exons 1 and 2. PCR products were subcloned, and transformants were transferred on several duplicate nylon membranes and grown on LB plates for use in a colony hybridization assay. Membranes were hybridized with oligonucleotide probes A and B (Table 1). Selective hybridization with probe A indicated the K6a isoform, while hybridization with probe B indicated the K6 b, c, d, e, or f isoform. These isoforms were discriminated by hybridization of duplicate filters with individual oligonucleotide probes C, D, E, F, and G (see Table 1). Optimal washing conditions for all the oligoprobes were determined using appropriate control DNAs. Under 0.5 × SSC and 42°C conditions, each probe was found to hybridize specifically with the expected purified isoform cDNA(s). Following autoradiography, each clone was scored for positive hybridization with oligonucleotide probes A-G. Hybridization with probe A indicated the K6a isoform; hybridization with probes D and F indicated either the K6b or K6f isoform; hybridization with probes C, E, and G indicated the K6c isoform; and hybridization with probes F and G indicated either the K6d or K6e isoform. Because our K6d genomic cloned lacked 198 bp at the 5′ end (see “Results”), we could not use probe C to distinguish it from the K6e isoform.Transient Expression of K6 IsoformsGenomic DNA fragments containing the entire coding sequence of the human K6a gene (~8-kbp EcoRI fragment), K6b (~18-kbp SalI fragment), and the K6c gene (~20-kbp SalI fragment) were subcloned into a cytomegalovirus vector (29Compton D.A. Cleveland D.W. J. Cell Biol. 1993; 120: 947-957Crossref PubMed Scopus (174) Google Scholar) containing a cytomegalovirus promoter-enhancer and a SV40 polyadenylation signal. Transient transfection assays were done in PtK2 epithelial cells (24Franke W.W. Schmid E. Osborn M. Weber K. Cytobiologie. 1978; 17: 391-411Google Scholar) cultured on glass coverslips, using the calcium-phosphate precipitation method (30Letai A. Coulombe P.A. Fuchs E. J. Cell Biol. 1992; 116: 1181-1195Crossref PubMed Scopus (137) Google Scholar). At 72 h post-transfection, cells were fixed in absolute methanol (−20°C, 15 min) and processed for indirect immunofluorescence (30Letai A. Coulombe P.A. Fuchs E. J. Cell Biol. 1992; 116: 1181-1195Crossref PubMed Scopus (137) Google Scholar). The primary antisera used were a rabbit polyclonal anti-K6 (17Stoler A. Kopan R. Duvic M. Fuchs E. J. Cell Biol. 1988; 107: 427-446Crossref PubMed Scopus (292) Google Scholar) and L2A1, a mouse monoclonal anti-K8/K18(31Chou C.F. Omary M.B. J. Biol. Chem. 1993; 268: 4465-4472Abstract Full Text PDF PubMed Google Scholar). Bound primary antibodies were detected with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Vector Labs), and a biotin-conjugated goat anti-mouse IgG (Kirkegaard and Perry Labs) followed by streptavidin-Texas Red conjugate (Vector Labs).RESULTSIsolation of Multiple Human Genes Encoding Keratin 6We first performed Southern blot analyses of human genomic DNA using probes derived from the previously characterized human K6b gene(21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar). When digested human DNAs were probed with a ~600-bp NcoI fragment corresponding to the coding portion of the human K6b gene exon 1, nearly 10 hybridization bands were apparent (data not shown). This NcoI fragment, however, also hybridized with the human K5 gene, even under stringent washing conditions. When a smaller (283 bp) NcoI-NarI fragment derived from the K6b gene exon 1 (which does not hybridize with the K5 gene) was used under stringent conditions (0.1 × SSC, 70°C), between three and five strong hybridization signals were detected in digested genomic DNAs from several randomly selected individuals (Fig. 1). These results suggested the existence of additional K6 isoform genes or K6-related gene(s) in the human genome.We screened a human genomic DNA library to isolate the human K6a gene as well as any other K6-encoding genes. A total of 1.5 × 106 clones were screened with various 32P-labeled cDNA probes derived from the coding sequence of the cloned human K6b gene(21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar). Over 100 clones were found to hybridize strongly under stringent conditions with a probe derived from K6b exon 1. Of these, 57 independent clones were isolated by repeated plaque purification, and analyzed by hybridization under stringent conditions with probes derived from the 3′ non-coding regions of the human K6b gene and K6a partial cDNA(21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar, 22Hanukoglu I. Fuchs E. Cell. 1983; 33: 915-924Abstract Full Text PDF PubMed Scopus (202) Google Scholar). This revealed that 30 clones hybridized with the K6a 3′ non-coding probe (group 1), 7 clones hybridized with the K6b 3′ non-coding probe (group 2), while the remaining 20 clones did not hybridize with either probe (group 3). Each group of genomic clones was further analyzed by digestion of purified phage DNAs and their hybridization with specific fragments from the K6b gene. Based on this analysis, the genomic clones in groups 1 and 3 could be further divided into three and four subgroup(s), respectively. Subsequently, hybridization-positive restriction fragments were isolated from representative clones in each of the 8 subgroups, subcloned into pBluescript SK(+), and analyzed by DNA sequencing. The sequence data obtained was compared with those reported for the human K6a partial cDNA and K6b gene(21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar, 22Hanukoglu I. Fuchs E. Cell. 1983; 33: 915-924Abstract Full Text PDF PubMed Scopus (202) Google Scholar).Group 1 clones, which hybridized to the K6a 3′ non-coding probe and included three restriction mapping subgroups, consisted of the human K6a gene and two novel K6-encoding genes, named K6c and K6d. Subsequent analyses indicated that these three genes shared virtually identical 3′ non-coding sequences (see below). Group 2 clones, which hybridized to the K6b 3′ non-coding probe, were independent isolates of the human K6b gene. The four subgroups identified among group 3 clones were found to include: (i) another potential K6-encoding gene (albeit partial); (ii) the K5 gene; and (iii) two full-length genes whose sequence display features characteristic of both the human K5 and K6 genes, designated K5/6-α and K5/6-β.Structural Organization of Keratin 6-Encoding Human Genes and Characterization of Their mRNAsWe completed the sequencing of the coding region, 5′- and 3′-flanking sequences and intron-exon junctions of the genomic clones potentially encoding K6-like proteins. Comparison of these sequences with the previously reported human K6b gene sequence (21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar) enabled us to locate all exons and define the 5′ regulatory sequences. Overlapping phage clones yielded full-length genes for K6a, K6b, and K6c, and restriction digestion/Southern blotting analyses of suitable phage clones allowed us to assign each of them to a specific hybridization product in human genomic DNAs processed in parallel (Fig. 1). The K6d genomic clone, on the other hand, lacked 198 bp at the 5′ end of exon 1, thus preventing assignment to a specific product in digested human genomic DNA. All K6 isoform genes except one are ~6-7 kbp long (Fig. 2) and thus are analogous to other known human type II keratin genes(21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar, 32Lersch R. Stellmach V. Stocks C. Giudice G. Fuchs E. Mol. Cell. Biol. 1989; 9: 3685-3697Crossref PubMed Scopus (71) Google Scholar, 33Johnson L.D. Idler W.W. Zhou X-M. Roop D.R. Steinert P.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1896-1900Crossref PubMed Scopus (109) Google Scholar, 34Krauss S. Franke W.W. Gene (Amst.). 1990; 86: 241-249Crossref PubMed Scopus (38) Google Scholar). In contrast, the K6c gene is remarkably long for a type II keratin gene, extending over 17 kbp. The K6 genes all contain nine exons interrupted by eight introns. The position of all eight introns (A-H) is identical in the K6a, K6c, and K6d genes and in the previously characterized human K6b, K7, and K5 genes(21Tyner A.L. Eichman M.J. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4683-4687Crossref PubMed Scopus (79) Google Scholar, 32Lersch R. Stellmach V. Stocks C. Giudice G. Fuchs E. Mol. Cell. Biol. 1989; 9: 3685-3697Crossref PubMed Scopus (71) Google Scholar, 35Glass C. Fuchs E. J. Cell Biol. 1988; 107: 1337-1350Crossref PubMed Scopus (37) Google Scholar). The introns are located within the protein-coding regions, and the sequences of the exon-intron boundaries conform to the consensus splicing signal(36Shapiro M.B. Senapathy P. Nucleic Acids Res. 1987; 15: 7155-7174Crossref PubMed Scopus (1956) Google Scholar). From restriction map" @default.
• W2034856955 created "2016-06-24" @default.
• W2034856955 creator A5027999029 @default.
• W2034856955 creator A5058634153 @default.
• W2034856955 creator A5074920093 @default.
• W2034856955 date "1995-08-01" @default.
• W2034856955 modified "2023-10-13" @default.
• W2034856955 title "Cloning and Characterization of Multiple Human Genes and cDNAs Encoding Highly Related Type II Keratin 6 Isoforms" @default.
• W2034856955 cites W1561949751 @default.
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